Methods of improving stress tolerance, growth and yield in plants

ABSTRACT

The present invention is directed to methods of improving yield in plants by applying an effective amount of a mixture of abscisic acid and malic acid to the plant. The present invention is further directed to methods of improving growth in plants by applying an effective amount of a mixture of abscisic acid and malic acid to the plant.

FIELD OF THE INVENTION

The present invention relates to methods of improving stress toleranceand yield in plants by applying an effective amount of a mixture of(S)-abscisic acid and malic acid to the plant. The present inventionfurther relates to methods of improving growth in plants by applying aneffective amount of a mixture of (S)-abscisic acid and malic acid to theplant.

BACKGROUND OF THE INVENTION

Growers continually attempt to grow the most productive crops possiblein order to maximize yields. Plant growth regulators are among the besttools that growers can use to influence the growth of plants based onthe restrictions of water and temperature. The effects of plant growthregulators on plants under different conditions can vary widely.Furthermore, it is difficult to predict the effect of simultaneouslyapplying more than one plant growth regulator to the plant.

(S)-abscisic acid (“ABA”) is an endogenous plant growth regulator withmany roles in growth and development. For example, ABA inhibits seedgermination by antagonizing gibberellins that stimulate the germinationof seeds. ABA promotes stress tolerance and maintains growth understress conditions (see Sharp R E et al. J Exp Bot, 2004 55:2343-2351).Interestingly, several studies have shown that maintaining ‘normal’ ABAlevels in well-watered plants is required to maintain shoot growth intomato (Sharp R E et al., J Exp Bot, 2000 51:1575-1584) and Arabidopsisthaliana (LeNoble M E et al. J Exp Bot, 2004 55:237-245). Moreover, ABAis responsible for the development and maintenance of dormancy in seedsand woody plants, which when deficient in ABA often demonstratepre-harvest sprouting of seeds due to a lack of dormancy induction.

Further, applications of ABA have also been shown to provide protectionfrom chilling and drought, as well as to increase the red color ofseedless table grapes. Examples of effective commercially available ABAformulations include ProTone™ and Contego™ (available from ValentBioSciences LLC).

Malic acid is an intermediate compound in the citric acid (TCA) cycle,and the C4 carbon fixation process of the chloroplast. In addition,malic acid is synthesized by stomatal guard cells in plant leaves andhas been shown to play an important role in stomatal control; however,it is unclear whether malic acid promotes opening or closure of thestomates (Araujo W L et al., Control of stomatal aperture, Plant SignalBehav. 2011 Sepember, 6(9), 1305-1311) as there are evidences supportingeach hypothesis.

Exogenous malic acid may promote plant growth (Talebi et al., Adv inAgri, 2014, 147: 278). Malic acid application resulted in increasedphotosynthesis under cadmium stress (Guo et al., Ecotoxicology andEnvironmental Safety, 141 (2017), 119-128). Thus, although malic acidhas an effect on growth and transpiration in plants; it is unclear howexogenous malic acid effects plant growth under water deficit stressconditions, especially in combination with ABA, a known stress tolerancecompound.

Accordingly, there is a need in the art for new methods to improve thegrowth of plants under abiotic stress conditions.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to methods of improvingstress tolerance in a plant comprising applying an effective amount of(S)-abscisic acid (“ABA”) and malic acid to the plant, wherein theweight ratio of ABA to malic acid is from about 3.3:1 to about 1:30.

In another aspect, the present invention is directed to methods ofimproving yield in a plant comprising applying an effective amount of(S)-abscisic acid (“ABA”) and malic acid to the plant, wherein theweight ratio of ABA to malic acid is from about 3.3:1 to about 1:33.3.

In another aspect, the present invention is directed to methods ofimproving plant growth comprising applying an effective amount of(S)-abscisic acid (“ABA”) and malic acid to the plant, wherein theweight ratio of ABA to malic acid is from about 3.3:1 to about 1:30.

DETAILED DESCRIPTION OF THE INVENTION

Applicant unexpectedly discovered a mixture of (S)-abscisic acid (“ABA”)and malic acid improved drought stress tolerance and plant growth understress conditions. Applicant further discovered that a mixture of ABAand malic acid unexpectedly improved plant growth under normalconditions. Further, the Applicant discovered that a mixture of ABA andmalic acid unexpectedly improved water use efficacy as demonstrated byunexpected increase in water banking (i.e. storing of water for futureuse). Applicant also discovered that a mixture of ABA and malic acidunexpectedly increased carbon fixing as demonstrated by an unexpectedincrease in photosynthetic rate and dry weight.

In one embodiment, the present invention is directed to methods ofimproving plant growth comprising applying an effective amount of ABAand malic acid to the plant, wherein the weight ratio of ABA to malicacid is from about 3.3:1 to about 1:33.3.

In another preferred embodiment, the plant in which plant growth isimproved is subject to an abiotic stress.

In another embodiment, the present invention is directed to methods ofimproving stress tolerance in a plant comprising applying an effectiveamount of ABA and malic acid to the plant, wherein the weight ratio ofABA to malic acid is from about 3.3:1 to about 1:30.

In a preferred embodiment, the stress tolerance that is improved is anabiotic stress.

In a preferred embodiment, ABA and malic acid are applied at a weightratio from about 10:1 to about 1:33.3, from about 10:1 to about 1:30:1,from about 3.3:1 to about 1:30, from about 3.3:1 to about 1:10, fromabout 3.3:1 to about 1:3.3, from about 3:1 to about 1:3, from about 1:3to about 1:33.3, from about 1:3 to about 1:30, from about 1:3 to about1:10, from about 3.3:1 to about 3:1 or about 3.3:1, 3:1, 1:1, 1:3,1:3.3, 1:10, 1:30 or 1:33.3.

In another embodiment, the plant is a monocotyledonous plant or adicotyledonous plant. In a preferred embodiment the plant is selectedfrom the group consisting of root, corm and tuber vegetable plants, bulbvegetable plants, leafy non-brassica vegetable plants, leafy brassicavegetable plants, succulent or dried legume plants, fruiting vegetableplants, cucurbit vegetable plants, citrus fruit plants, pome fruitplants, stone fruit plants, berry and small fruit plants, tree nutplants, cereal crops, forage and fodder grasses and hay, non-grassanimal feed plants, herb plants, spice plants, flower plants, beddingplants, ornamental flower plants, artichoke, asparagus, tropical fruitplants, hops, malanga, peanut, pomegranate plants, oil seed vegetableplants, tobacco plants, turf grass and watercress plant. In a morepreferred embodiment, the plant is wheat, corn, rice or lettuce.

In a preferred embodiment, the root, corm and tuber vegetable plants areselected from the group consisting of arracacha, arrowroot, Chineseartichoke, Jerusalem artichoke, garden beet, sugar beet, edible burdock,edible canna, carrot, bitter cassava, sweet cassava, celeriac, rootchayote, turnip-rooted chervil, chicory, chufa, dasheen (taro), ginger,ginseng, horseradish, leren, turnip-rooted parsley, parsnip, potato,radish, oriental radish, rutabaga, salsify, black salsify, Spanishsalsify, skirret, sweet potato, tanier, turmeric, turnip, yam bean, trueyam, and cultivars, varieties and hybrids thereof.

In another preferred embodiment, the bulb vegetable plants are selectedfrom the group consisting of fresh chive leaves, fresh Chinese chiveleaves, bulb daylily, elegans hosta, bulb fritillaria, fritillarialeaves, bulb garlic, great-headed bulb garlic, serpent bulb garlic,kurrat, lady's leek, leek, wild leek, bulb lily, Beltsville bunchingonion, bulb onion, Chinese bulb onion, fresh onion, green onion,macrostem onion, pearl onion, potato bulb onion, potato bulb, tree oniontops, Welsh onion tops, bulb shallot, fresh shallot leaves, andcultivars, varieties and hybrids thereof.

In a further embodiment, the leafy non-brassica vegetable plants areselected from the group consisting of Chinese spinach Amaranth, leafyAmaranth, arugula (roquette), cardoon, celery, Chinese celery, celtuce,chervil, Chinese spinach, edible-leaved chrysanthemum, garlandchrysanthemum, corn salad, garden cress, upland cress, dandelion,dandelion leaves, sorrels (dock), endive (escarole), Florence fennel,head lettuce, leaf lettuce, orach, parsley, garden purslane, winterpurslane, radicchio (red chicory), rhubarb, spinach, New Zealandspinach, vine spinach, Swiss chard, Tampala, and cultivars, varietiesand hybrids thereof.

In another embodiment, the leafy brassica vegetable plants are selectedfrom the group consisting of broccoli, Chinese broccoli (gai lon),broccoli rabe (rapini), Brussels sprouts, cabbage, Chinese cabbage (bokchoy), Chinese napa cabbage, Chinese mustard cabbage (gai choy),cauliflower, cavolo broccoli, collards, kale, kohlrabi, mizuna, mustardgreens, mustard spinach, rape greens, turnip greens and cultivars,varieties and hybrids thereof. In yet another embodiment, the succulentor dried vegetable legumes are selected from the group consisting ofLupinusbeans, Phaseolusbeans, Vignabeans, broad beans (fava), chickpea(garbanzo), guar, jackbean, lablab bean, lentil, Pisumpeas, pigeon pea,soybean, immature seed soybean, sword bean, peanut, and cultivars,varieties and hybrids thereof. In a preferred embodiment, theLupinusbeans include grain lupin, sweet lupin, white lupin, white sweetlupin, and hybrids thereof. In another preferred embodiment, thePhaseolusbeans include field bean, kidney bean, lima bean, navy bean,pinto bean, runner bean, snap bean, tepary bean, wax bean, and hybridsthereof. In yet another preferred embodiment, the Vignabeans includeadzuki bean, asparagus bean, blackeyed bean, catjang, Chinese longbean,cowpea, Crowder pea, moth bean, mung bean, rice bean, southern pea, urdbean, yardlong bean, and hybrids thereof. In another embodiment, thePisumpeas include dwarf pea, edible-podded pea, English pea, field pea,garden pea, green pea, snow pea, sugar snap pea, and hybrids thereof. Ina preferred embodiment, the dried vegetable legume is soybean. In a morepreferred embodiment, the dried vegetable legume is genetically modifiedsoybean.

In a further embodiment, the fruiting vegetable plants are selected fromthe group consisting of bush tomato, cocona, currant tomato, gardenhuckleberry, goji berry, groundcherry, martynia, naranjilla, okra, peaeggplant, pepino, bell peppers, non-bell peppers, roselle, eggplant,scarlet eggplant, African eggplant, sunberry, tomatillo, tomato, treetomato, and cultivars, varieties and hybrids thereof. In a preferredembodiment, the peppers include bell peppers, chili pepper, cookingpepper, pimento, sweet peppers, and hybrids thereof.

In an embodiment, the cucurbit vegetable plants are selected from thegroup consisting of Chayote, Chayote fruit, waxgourd (Chinese preservingmelon), citron melon, cucumber, gherkin, edible gourds,Momordicaspecies, muskmelons, pumpkins, summer squashes, wintersquashes, watermelon, and cultivars, varieties and hybrids thereof. In apreferred embodiment, edible gourds include hyotan, cucuzza, hechima,Chinese okra, and hybrids thereof. In another preferred embodiment, theMomordicavegetables include balsam apple, balsam pear, bittermelon,Chinese cucumber, and hybrids thereof. In another preferred embodiment,the muskmelon include true cantaloupe, cantaloupe, casaba, crenshawmelon, golden pershaw melon, honeydew melon, honey balls, mango melon,Persian melon, pineapple melon, Santa Claus melon, snake melon, andhybrids thereof. In yet another preferred embodiment, the summer squashinclude crookneck squash, scallop squash, straightneck squash, vegetablemarrow, zucchini, and hybrids thereof. In a further preferredembodiment, the winter squash includes butternut squash, calabaza,hubbard squash, acorn squash, spaghetti squash, and hybrids thereof.

In another embodiment, the citrus fruit plants are selected from thegroup consisting of limes, calamondin, citron, grapefruit, Japanesesummer grapefruit, kumquat, lemons, Mediterranean mandarin, sour orange,sweet orange, pummelo, Satsuma mandarin, tachibana orange, tangelo,mandarin tangerine, tangor, trifoliate orange, uniq fruit, andcultivars, varieties and hybrids thereof. In a preferred embodiment, thelimes are selected from the group consisting of Australian desert lime,Australian finger lime, Australian round lime, Brown River finger lime,mount white lime, New Guinea wild lime, sweet lime, Russell River lime,Tahiti lime, and hybrids thereof.

In an embodiment, the pome fruit plants are selected from the groupconsisting of apple, azarole, crabapple, loquat, mayhaw, medlar, pear,Asian pear, quince, Chinese quince, Japanese quince, tejocote, andcultivars, varieties and hybrids thereof.

In another embodiment, the stone fruit plants are selected from thegroup consisting of apricot, sweet cherry, tart cherry, nectarine,peach, plum, Chicksaw plum, Damson plum, Japanese plum, plumcot, freshprune, and cultivars, varieties and hybrids thereof.

In a further embodiment, the berries and small fruit plants are selectedfrom the group consisting of Amur river grape, aronia berry, bayberry,bearberry, bilberry, blackberry, blueberry, lowbush blueberry, highbushblueberry, buffalo currant, buffaloberry, che, Chilean guava,chokecherry, cloudberry, cranberry, highbush cranberry, black currant,red currant, elderberry, European barberry, gooseberry, grape, ediblehoneysuckle, huckleberry, jostaberry, Juneberry (Saskatoon berry),lingonberry, maypop, mountain pepper berries, mulberry, muntries, nativecurrant, partridgeberry, phalsa, pincherry, black raspberry, redraspberry, riberry, salal, sea buckthorn, serviceberry, strawberry, wildraspberry, and cultivars, varieties and hybrids thereof. In a preferredembodiment, the blackberries include Andean blackberry, arcticblackberry, bingleberry, black satin berry, boysenberry, brombeere,California blackberry, Chesterberry, Cherokee blackberry, Cheyenneblackberry, common blackberry, coryberry, darrowberry, dewberry, Dirksenthornless berry, evergreen blackberry, Himalayaberry, hullberry,lavacaberry, loganberry, lowberry, Lucreliaberry, mammoth blackberry,marionberry, mora, mures deronce, nectarberry, Northern dewberry,olallieberry, Oregon evergreen berry, phenomenalberry, rangeberry,ravenberry, rossberry, Shawnee blackberry, Southern dewberry, tayberry,youngberry, zarzamora, and hybrids thereof.

In another embodiment, the tree nut plants are selected from the groupconsisting of almond, beech nut, Brazil nut, Brazilian pine, bunya,butternut, bur oak, Cajou nut, candlenut, cashew, chestnut, chinquapin,coconut, coquito nut, dika nut, gingko, Guiana chestnut, hazelnut(filbert), heartnut, hickory nut, Japanese horse-chestnut, macadamianut, mongongo nut, monkey-pot, monkey puzzule nut, Okari nut, Pachiranut, peach palm nut, pecan, Pili nut, pistachio, Sapucaia nut, tropicalalmond, black walnut, English walnut, yellowhorn, and cultivars,varieties and hybrids thereof.

In a further embodiment, the cereal grains are selected from the groupconsisting of barley, buckwheat, pearl millet, proso millet, oats, corn,field corn, sweet corn, seed corn, popcorn, rice, rye, sorghum (milo),sorghum species, grain sorghum, sudangrass (seed), teosinte, triticale,wheat, wild rice, and cultivars, varieties and hybrids thereof. In apreferred embodiment, the cereal grain is selected from the groupconsisting of wheat, rice and corn. In a more preferred embodiment, thecereal grain is genetically modified corn.

In yet another embodiment, the grass forage, fodder and hay are selectedfrom the group consisting of grasses that are members of the Gramineaefamily except sugarcane and those species included in the cereal grainsgroup, pasture and range grasses, and grasses grown for hay or silage.In further embodiments, the Gramineae grasses may be green or cured.

In an embodiment, the non-grass animal feeds are selected from the groupconsisting of alfalfa, velvet bean, trifolium clover, melilotus clover,kudzu, lespedeza, lupin, sainfoin, trefoil, vetch, crown vetch, milkvetch, and cultivars, varieties and hybrids thereof.

In another embodiment, the herbs and spice plants are selected from thegroup consisting of allspice, angelica, anise, anise seed, star anise,annatto seed, balm, basil, borage, burnet, chamomile, caper buds,caraway, black caraway, cardamom, cassia bark, cassia buds, catnip,celery seed, chervil, chive, Chinese chive, cinnamon, clary, clove buds,coriander leaf, coriander seed, costmary, culantro leaves, culantroseed, cilantro leaves, cilantro seed, cumin, dillweed, dill seed,fennel, common fennel, Florence fennel seed, fenugreek, grains ofparadise, horehound, hyssop, juniper berry, lavender, lemongrass, leaflovage, seed lovage, mace, marigold, marjoram, mint, mustard seed,nasturtium, nutmeg, parsley, pennyroyal, black pepper, white pepper,poppy seed, rosemary, rue, saffron, sage, summer savory, winter savory,sweet bay, tansy, tarragon, thyme, vanilla, wintergreen, woodruff,wormwood, and cultivars, varieties and hybrids thereof. In a preferredembodiment, the mints are selected from the group consisting ofspearmint, peppermint, and hybrids thereof.

In yet another embodiment, artichokes are selected from the groupconsisting of Chinese artichoke, Jerusalem artichoke, and cultivars,varieties and hybrids thereof.

In an embodiment, the tropical fruit plants are selected from the groupconsisting of anonna, avocado, fuzzy kiwifruit, hardy kiwifruit, banana,plantain, caimito, carambola (star fruit), guava, longan, sapodilla,papaya, passion fruit, mango, lychee, jackfruit, dragon fruit, mameysapote, coconut cherimoya, canistrel, monstera, wax jambu, pomegranate,rambutan, pulasan, Pakistani mulberry, langsat, chempedak, durian, figpineapple, jaboticaba, mountain apples, and cultivars, varieties andhybrids thereof. In a further embodiment, the oil seed vegetable plantsare selected from the group consisting of borage, calendula, castor oilplant, tallowtree, cottonseed, crambe, cuphea, echium, euphorbia,evening primrose, flax seed, gold of pleasure, hare's ear, mustard,jojoba, lesquerella, lunaria, meadowfoam, milkweed, niger seed, oilradish, poppy seed, rosehip, sesame, stokes aster, sweet rocket,tallowwood, tea oil plant, vermonia, canola, or oil rapeseed, safflower,sunflower, and cultivars, varieties and hybrids thereof.

In another embodiment, the plant is subjected to drought stress. As usedherein, “drought stress” refers to watering conditions wherein plantgrowth is significantly slowed as compared to those where wateravailability is sufficient to support optimal growth and development.

In a preferred embodiment, ABA and malic acid is applied prior to orduring the advent of abiotic stress. When the intended stress isdrought, application of ABA and malic acid occurs prior to or duringdrought stress. Application prior to drought allows for banking of soilwater. By conserving soil water plants can extend survival and growthduring critical growth stages, when yield losses due to water stress arehigher.

In another preferred embodiment, from about 1 to 1,000 parts per million(“ppm”) of ABA are applied to the plant, more preferably from about 30to 1,000 ppm or from 30 to 300 ppm.

In another preferred embodiment, from about 1 to 1,000 parts per million(“ppm”) of malic acid are applied to the plant, more preferably fromabout 30 to 1,000 ppm or from 30 to 300 ppm.

In another preferred embodiment, ABA is applied to the plant at a ratefrom about 1 to about 1,000 liters per hectare (“L/Ha”), more preferablyfrom about 10 to about 500 L/Ha and most preferably from about 100 toabout 200 L/Ha.

In another preferred embodiment, malic acid is applied to the plant at arate from about 1 to about 1,000 L/Ha, more preferably from about 10 toabout 500 L/Ha and most preferably from about 100 to about 200 L/Ha.

The ABA and malic acid mixture can be applied by any convenient means.Those skilled in the art are familiar with the modes of application thatinclude foliar applications such as spraying, dusting, and granularapplications; soil applications including spraying, in-furrowtreatments, or side-dressing.

In another preferred embodiment, the present invention is directed to acomposition comprising ABA and malic acid, wherein the weight ratio ofABA to malic acid is from about 10:1 to about 1:33.3, from about 10:1 toabout 1:30:1, from about 3.3:1 to about 1:30, from about 3.3:1 to about1:10, from about 3.3:1 to about 1:3.3, from about 3:1 to about 1:3, fromabout 1:3 to about 1:33.3, from about 1:3 to about 1:30, from about 1:3to about 1:10, from about 3.3:1 to about 3:1 or about 3.3:1, 3:1, 1:1,1:3, 1:3.3, 1:10, 1:30 or 1:33.3.

Aqueous spray solutions utilized in the present invention generallycontain from about 0.01% to about 0.5% (v/v) of a non-ionicsurface-active agent.

The surface-active agent comprises at least one non-ionic surfactant. Ingeneral, the non-ionic surfactant may be any known non-ionic surfactantin the art. Suitable non-ionic surfactants are in general oligomers andpolymers. Suitable polymers include alkyleneoxide random and blockcopolymers such as ethylene oxide-propylene oxide block copolymers(EO/PO block copolymers), including both EO-PO-EO and PO-EO-PO blockcopolymers; ethylene oxide-butylene oxide random and block copolymers,C2-6 alkyl adducts of ethylene oxide-propylene oxide random and blockcopolymers, C2-6 alkyl adducts of ethylene oxide-butylene oxide randomand block copolymers, polyoxyethylene-polyoxypropylene monoalkylethers,such as methyl ether, ethyl ether, propyl ether, butyl ether or mixturesthereof; vinylacetate/vinylpyrrolidone copolymers; alkylatedvinylpyrrolidone copolymers; polyvinylpyrrolidone; andpolyalkyleneglycol, including the polypropylene glycols and polyethyleneglycols. Other non-ionic agents are the lecithins; and silicone surfaceactive agents (water soluble or dispersible surface-active agents havinga skeleton which comprises a siloxane chain e.g. Silwet L77®). Asuitable mixture in mineral oil is ATPLUS® 411.

As used herein, “effective amount” refers to the amount of the ABAand/or malic acid that will improve growth, drought stress tolerance,and/or yield. The “effective amount” will vary depending on the ABA andmalic acid concentrations, the plant species or variety being treated,the severity of the stress, the result desired, and the life stage ofthe plants, among other factors. Thus, it is not always possible tospecify an exact “effective amount.” However, an appropriate “effectiveamount” in any individual case may be determined by one of ordinaryskill in the art.

As used herein, “improving” means that the plant has more of the qualitythan the plant would have had it if it had not been treated by methodsof the present invention.

As used herein, all numerical values relating to amounts, weightpercentages and the like are defined as “about” or “approximately” eachparticular value, namely, plus or minus 10% (±10%). For example, thephrase “at least 5% by weight” is to be understood as “at least 4.5% to5.5% by weight.” Therefore, amounts within 10% of the claimed values areencompassed by the scope of the claims.

The articles “a,” “an” and “the” are intended to include the plural aswell as the singular, unless the context clearly indicates otherwise.

The disclosed embodiments are simply exemplary embodiments of theinventive concepts disclosed herein and should not be considered aslimiting, unless the claims expressly state otherwise.

The following examples are intended to illustrate the present inventionand to teach one of ordinary skill in the art how to use theformulations of the invention. They are not intended to be limiting inany way.

EXAMPLES Example 1 Increased Stress Tolerance in Cucumber Plants UnderDrought Stress

10 sets of cucumber plants (n=5) were each treated on 10 post-plantingday (“DAP”) with either 30 or 100 ppm ABA, 30 or 100 ppm malic acid ormixtures thereof. Water was withheld starting on 10 DAP for 5 sets andthe other sets were fully irrigated. Green leaf area was measured usinga handheld Greenseeker® crop sensor, which uses a normalized differencevegetative index (“NDVI”) to measure green leaf area. Green leaf areawas measured and recorded everyday starting on 10 DAP and ending on day4 post-treatment (“DAT”). Results of these measurement can be seen inTable 1, below.

To determine if the mixtures provided unexpected results, the observedcombined efficacy (“OCE”) was divided by the expected combined efficacy(“ECE”) to give an OCE/ECE ratio wherein the expected ECE is calculatedby the Abbott method:

ECE=A+B−(AB/100),

wherein ECE is the expected combined efficacy and in which A and B arethe efficacy provided by the single active ingredients. If the ratiobetween the OCE of the mixture and the ECE of the mixture is greaterthan 1, then greater than expected interactions are present in themixture. (Gisi, The American Phytopathological Society, 86:11,1273-1279,1996).

TABLE 1 % Change OCE/ECE NDVI from STC ratio STC 0.595 n/a n/a ABA 100ppm 0.72 21.0% n/a Malic acid 30 ppm 0.563 −5.4% n/a ABA 100 ppm + 0.94458.7% 1.4 Malic acid 30 ppm ABA 100 ppm + 0.956 60.7% n/a Malic acid 100ppm “STC” denotes surfactant treated control

As seen in Table 1, ABA increased green leaf area whereas malic aciddecreased green leaf area. A mixture of ABA and malic acid at a ratio of3.3:1 demonstrated an unexpected increase in green leaf area.

Example 2 Increased Stress Tolerance in Cucumber Plants Under DroughtStress

11 sets of cucumber plants (n=8) were each treated on day 10 DAP witheither 30 or 100 ppm ABA, 30 or 100 ppm malic acid or mixtures thereof.Water was withheld starting on 10 DAP. Green leaf area was measured andrecorded everyday starting on 10 DAP and ending on day 4 post-treatment(“DAT”). Results of these measurement can be seen in Table 2, below.

TABLE 2 % Change OCE/ECE NDVI from UTC ratio STC 1.58   0% n/a ABA 30ppm 1.32 −16.5%  n/a ABA 100 ppm 1.78 12.7% n/a Malic acid 30 ppm 1.7812.7% n/a Malic acid 100 ppm 1.36 −13.9%  n/a ABA 30 ppm + 2.07 31.0%1.4 Malic acid 30 ppm ABA 30 ppm + 2.23 41.1% 2.0 Malic acid 100 ppm ABA100 ppm + 2.02 27.8% 1.0 Malic acid 30 ppm ABA 100 ppm + 2.14 35.4% 1.4Malic acid 100 ppm “STC” denotes surfactant treated control

As seen in Table 2, ABA and malic acid each increased and decreasedgreen leaf area depending on concentration. Unexpectedly, a mixture ofABA and malic acid at ratios of 1:1, 1:3.3, 3.3:1 provided greater thanexpected increase in green leaf area when plants were subjected to waterdeficit stress.

Example 3 Increased Dry Weight in Cucumber Plants Under Drought Stress

6 sets of cucumber plants (n=5) were each treated on 10 DAP with either100 ppm ABA, 30, 100 or 300 ppm malic acid or mixtures thereof. Waterwas withheld from 10 DAP to 4 DAT. Water was applied on 4 DAT. Water waswithheld from 5 DAT to 7 DAT. Plants were harvested, and dry weight wasmeasured and recorded on 7 DAT. This experiment was repeated withharvest occurring 8 DAT. Results of these measurements can be seen inTables 3 and 4, below, respectively.

TABLE 3 Increase OCE/ECE in Dry % Change ratio Weight from UTC (Linear)STC 0.99 n/a n/a ABA 100 ppm 1.04 5.1% n/a Malic acid 30 ppm 1.01 2.0%n/a ABA 100 ppm + 1.24 25.3% 1.2 Malic acid 30 ppm ABA 100 ppm + 1.3637.4% n/a Malic acid 100 ppm ABA 100 ppm + 1.13 14.1% n/a Malic acid 300ppm “STC” denotes—surfactant treated control

TABLE 4 Increase in Dry % Change OCE/ECE Treatment Weight from UTC ratioSTC 0.98 0.0% n/a ABA 100 ppm 0.96 −2.2% n/a Malic acid 30 ppm 1.1415.9% n/a ABA 100 ppm + 1.27 30.0% 1.1 Malic acid 30 ppm ABA 100 ppm +1.04 6.1% n/a Malic acid 100 ppm ABA 100 ppm + 0.97 −1.4% n/a Malic acid300 ppm “STC” denotes—surfactant treated control

As seen in Table 3 and 4, mixtures of ABA and malic acid improved dryweight over the control and over the application of either alone at allconcentrations. The mixtures of ABA and malic acid at a 3.3:1 ratiodemonstrated unexpected increase in dry weight.

Example 4 Increased Water Banking in Wheat Plants Under Drought Stress

8 sets of wheat plants (n=8) were each treated 1 week after anthesiswith either 300 ppm ABA, 1000 ppm malic acid or a mixture thereof. Waterwas withheld for three days after treatment and kept well-watered fornext four days. Chemical spray treatment was repeated one week after theinitial spray followed by similar drought cycle and irrigation. Thisexperiment was then repeated. Evapotranspiration (i.e. change in potweight) was measured on 1, 2 and 3 DAT for each cycle. Results showingunexpected increase in water banking via application of ABA and malicacid can be seen for the 2^(nd) cycle of the 1^(st) experiment and forthe 1^(st) and 2^(nd) cycles of the 2^(nd) experiment in Tables 5-7,below.

TABLE 5 Experiment #1 Evapotranspiration % Change from UTC OCE/ECE ratio(2^(nd) Cycle) 1 DAT 2 DAT 3 DAT 1 DAT 2 DAT 3 DAT 1 DAT 2 DAT 3 DAT STC70.18 83.62 22.46 0 0 0 n/a n/a n/a ABA 300 ppm 46.62 83.36 34.73 −33.6%−0.3% 54.6% n/a n/a n/a Malic acid 1000 ppm 74.37 82.09 22.72 6.0% −1.8%1.2% n/a n/a n/a ABA 300 ppm + 50.04 74.45 40.59 −28.7% −11.0% 80.7% 1.00.9 1.2 Malic acid 1000 ppm

TABLE 6 Experiment #2 Evapotranspiration % Change from UTC OCE/ECE ratio(1^(st) Cycle) 1 DAT 2 DAT 3 DAT 1 DAT 2 DAT 3 DAT 1 DAT 2 DAT 3 DAT STC111.15 61.76 18.17 0.0% 0.0% 0.0% n/a n/a n/a ABA 300 ppm 61.61 67.9144.98 −44.6% 10.0% 147.6% n/a n/a n/a Malic acid 1000 ppm 102.06 67.8318.18 −8.2% 9.8% 0.1% n/a n/a n/a ABA 300 ppm + 55.2 67.42 51.26 −50.3%9.2% 182.1% 1.1 0.9 1.1 Malic acid 1000 ppm

TABLE 7 Experiment #2 Evapotranspiration % Change from UTC OCE/ECE ratio(2^(nd) Cycle) 1 DAT 2 DAT 3 DAT 1 DAT 2 DAT 3 DAT 1 DAT 2 DAT 3 DAT STC50.81 60.12 43.2 0.0% 0.0% 0.0% n/a n/a n/a ABA 300 ppm 57.27 71.8346.86 12.7% 19.5% 8.5% n/a n/a n/a Malic acid 1000 ppm 51.41 62.73 46.171.2% 4.3% 6.9% n/a n/a n/a ABA 300 ppm + 50.41 65.81 57.85 −0.8% 9.5%33.9% 0.9 0.9 1.2 Malic acid 1000 ppm“STC” denotes untreated control

As seen in Tables 5-7, both ABA and malic acid alone demonstratedevidence of water banking. Evidence of water banking can be seen by thegreater amounts of evapotranspiration during drought stress,particularly 3 DAT. A mixture of ABA and malic acid at a ratio of 1:3.3demonstrated unexpected levels of water banking, especially 3 DAT during1^(st) and 2^(nd) cycle of drought stress.

Example 5 Increased Water Banking in Wheat Plants Under Drought Stress

10 sets of wheat plants (n=6) were each treated 1 week after anthesiswith either 100 or 300 ppm ABA, 100, 300 or 1000 ppm malic acid ormixtures thereof in a 0.025% Latron B 1956® (available from J.R. SimplotCompany) surfactant solution. Water was withheld from the day ofchemical treatment. Evapotranspiration was measured on 1, 2 and 3 DAT asthe amount of water left in the pot compared to day 0. Results can beseen in Table 8, below.

TABLE 8 Evapotranspiration % Change from STC OCE/ECE ratio 1 DAT 2 DAT 3DAT 1 DAT 2 DAT 3 DAT 1 DAT 2 DAT 3 DAT STC 0.763 0.404 0.257 0.0% 0.0%0.0% n/a n/a n/a 100 ppm ABA 0.834 0.582 0.321 9.4% 44.2% 24.7% n/a n/an/a 300 ppm ABA 0.837 0.610 0.349 9.8% 51.1% 35.5% n/a n/a n/a 100 ppmMalic acid 0.780 0.438 0.269 2.3% 8.4% 4.5% n/a n/a n/a 300 ppm Malicacid 0.751 0.404 0.260 −1.5% −0.1% 1.1% n/a n/a n/a 1000 ppm Malic acid0.736 0.390 0.251 −3.4% −3.4% −2.4% n/a n/a n/a 100 ppm ABA + 0.8540.617 0.344 12.0% 52.9% 33.6% 1.0 1.0 1.0 100 ppm Malic acid 100 ppmABA + 0.850 0.620 0.346 11.5% 53.7% 34.4% 1.0 1.1 1.1 300 ppm Malic acid100 ppm ABA + 0.859 0.631 0.350 12.7% 56.2% 36.0% 1.1 1.1 1.1 1000 ppmMalic acid 300 ppm ABA + 0.876 0.658 0.386 14.9% 63.1% 49.8% 1.0 1.0 1.1100 ppm Malic acid 300 ppm ABA + 0.880 0.675 0.407 15.4% 67.2% 57.9% 1.11.1 1.2 300 ppm Malic acid 300 ppm ABA + 0.889 0.685 0.413 16.5% 69.8%60.3% 1.1 1.1 1.2 1000 ppm Malic acid “STC” denotes surfactant treatedcontrol

As seen in Table 8, both ABA and malic acid alone demonstrated evidenceof water banking. Evidence of water banking can be seen by the greateramounts of evapotranspiration during drought stress, particularly 3 DAT.A mixture of ABA and malic acid at a ratio of 3:1, 1:1, 1:3, 1:3.3, and1:10 demonstrated unexpected levels of water banking, especially 3 DATas the amount of water left in the pot compared to day 0.

Example 6 Increased Water Banking in Wheat Plants Under Drought Stress

−10 sets of wheat plants (n=6) were each treated 1 week after anthesiswith either 100 or 300 ppm ABA, 1000 ppm malic acid or mixtures thereofin a 0.025% Latron B 1956® surfactant solution. Water was withheldduring the treatment. Evapotranspiration was measured on 1, 2 and 3 DAT.Results can be seen in Table 9, below.

TABLE 9 Evapotranspiration % Change from STC OCE/ECE ratio 1 DAT 2 DAT 3DAT 1 DAT 2 DAT 3 DAT 1 DAT 2 DAT 3 DAT STC 0.728 0.358 0.230 7.8% 42.6%15.5% n/a n/a n/a 100 ppm ABA 0.785 0.510 0.266 9.9% 56.1% 26.6% n/a n/an/a 300 ppm ABA 0.800 0.558 0.292 0.6% 3.6% 3.6% n/a n/a n/a 1000 ppmMalic acid 0.733 0.370 0.239 16.6% 78.0% 63.8% n/a n/a n/a 300 ppm ABA +0.848 0.636 0.378 7.8% 42.6% 15.5% 1.1 1.1 1.3 1000 ppm Malic acid “STC”denotes surfactant treated control

As seen in Table 9, both ABA and malic acid alone demonstrated evidenceof water banking. Evidence of water banking can be seen by the greateramounts of evapotranspiration during drought stress, particularly 3 DAT.A mixture of ABA and malic acid at a ratio of 1:3.3 demonstratedunexpected levels of water banking, especially 3 DAT as the amount ofwater left in the pot compared to day 0.

Example 7 Increased Grain Yield in Wheat Plants Under Drought Stress

8 sets of wheat plants (n=8) were each treated at one week afteranthesis with either 300 ppm ABA, 1000 ppm malic acid or a mixturethereof; chemical treatment with same compounds was repeated one weekafter initial spray. Water was withheld during the treatment. Shootweight, spike weight and grain yield were measured at physiologicalmaturity. Results can be seen in Table 10, below.

TABLE 10 Drought Stress % Change from UTC OCE/ECE ratio Shoot SpikeGrain Shoot Spike Grain Shoot Spike Grain Weight Weight Weight WeightWeight Weight Weight Weight Weight STC 3.03 6.80 4.99 0.0% 0.0% 0.0% n/an/a n/a ABA 300 ppm 3.38 7.27 5.25 11.9% 6.8% 5.1% n/a n/a n/a Malicacid 1000 ppm 3.13 6.98 4.91 3.5% 2.6% −1.7% n/a n/a n/a ABA 300 ppm +3.39 7.61 5.58 11.9% 11.9% 11.8% 1.0 1.0 1.1 Malic acid 1000 ppm “STC”denotes surfactant treated control

As can be seen in Table 10, ABA alone demonstrated evidence of increasedgrain weight, whereas malic acid alone demonstrated evidence ofdecreased grain weight. A mixture of ABA and malic acid at a 1:3.3 ratiodemonstrated an unexpected increase in grain weight.

Example 8 Increased Weight in Lettuce Under Drought Stress

8 sets of lettuce plants (n=8) were each treated 20 DAP with either 300ppm ABA, 1000 ppm malic acid or a mixture thereof. Water was withheldduring the treatment. Fresh weight and dry weight were measured 34 DAT.Results can be seen in Tables 11 and 12, below.

TABLE 11 Weight % Change OCE/ECE Fresh Weight (g) from UTC ratio UTC15.3 n/a n/a ABA 300 ppm 15.9 6.7% n/a Malic acid 1000 ppm 10.8 −26.7%n/a ABA 300 ppm + 17.8 16.7% 1.5 Malic acid 1000 ppm

TABLE 12 Weight % Change OCE/ECE Dry Weight (g) from UTC ratio UTC 1.88n/a n/a ABA 300 ppm 1.75 −5.6% n/a Malic acid 1000 ppm 1.50 −16.7% n/aABA 300 ppm + 2.15 22.2% 1.6 Malic acid 1000 ppm“UTC” denotes untreated control

As can be seen in Tables 11 and 12, ABA alone demonstrated evidence ofincreased fresh weight, whereas ABA alone demonstrated evidence ofdecreased dry weight and malic acid alone demonstrated evidence ofdecreased fresh and dry weight. A mixture of ABA and malic acid at a1:3.3 ratio demonstrated an unexpected increase in both fresh weight anddry weight.

Example 9 Increased Photosynthesis Rate under Drought Stress

Seven sets of corn plants (n=7) were each treated 16 DAP with either 300or 1000 ppm ABA, 1000 ppm malic acid or mixtures thereof. Water waswithheld from the date of chemical treatment. Photosynthesis rate wasmeasured 1, 4 and 6 DAT. This experiment was repeated.

Results can be seen in Tables 13 and 14, below.

TABLE 13 Photosynthesis Rate CO₂ assimilation (umol m⁻² s⁻¹) % Changefrom UTC OCE/ECE ratio Experiment #1 1 DAT 4 DAT 6 DAT 1 DAT 4 DAT 6 DAT1 DAT 4 DAT 6 DAT STC 23.91 21.29 4.52 0.0% 0.0% 0.0% n/a n/a n/a 300ppm ABA 12.93 20.51 9.14 −45.8% −4.8% 125.0% n/a n/a n/a 1000 ppm ABA9.23 19.05 19.15 −62.5% −14.3% 375.0% n/a n/a n/a 1000 ppm Malic 24.1921.84 5.36 0.0% 4.8% 25.0% n/a n/a n/a acid 300 ppm ABA + 14.73 22.4811.38 −41.7% 9.5% 200.0% 1.1 1.1 1.2 1000 ppm Malic acid 1000 ppm ABA +7.74 16.51 15.03 −70.8% −23.8% 275.0% 0.8 0.8 0.8 1000 ppm Malic acid

TABLE 14 Photosynthesis Rate % Change from UTC OCE/ECE ratio Experiment#2 1 DAT 4 DAT 6 DAT 1 DAT 4 DAT 6 DAT 1 DAT 4 DAT 6 DAT STC 22.78 19.839.99 0.0% 0.0% 0.0% n/a n/a n/a 300 ppm ABA 18.23 20.06 9.21 −17.4% 0.0%−10.0% n/a n/a n/a 1000 ppm ABA 16.55 21.14 12.61 −30.4% 5.0% 30.0% n/an/a n/a 1000 ppm Malic 21.97 21.05 8.09 −4.3% 5.0% −20.0% n/a n/a n/aacid 300 ppm ABA + 17.01 21.29 13.10 −26.1% 10.0% 40.0% 0.9 1.0 2.0 1000ppm Malic acid 1000 ppm ABA + 15.36 20.31 11.55 −34.8% 0.0% 20.0% 1.00.9 1.1 1000 ppm Malic acid “UTC” denotes surfactant treated control“UTC” denotes surfactant treated control

As can be seen in Tables 13 and 14, ABA alone demonstrated evidence ofincreased photosynthetic rate at 6 DAT and malic acid alone demonstratedevidence of both increased and decreased photosynthetic rates at 6 DAT.A mixture of ABA and malic acid at a 1:3.3 and a 1:1 ratio demonstratedan unexpected increase in photosynthetic rate at 6 DAT.

Example 10 Increased Yield of Rice Plants Under Not-Stressed Conditionsand Drought Stressed Conditions

A commercial semi-dwarf rice plant was used to test whether thecombination of ABA and malic acid improves grain yield more than eitheralone. Rice was grown in the greenhouse using media composed of ProfileGreens Grade in combination with ProMix®-BX in pots, which weresaturated with water and fertilizer solutions. Treatments were appliedto rice plants at early grain filling stage (5-20 days post-anthesis).Unexpected increases in grain yields were observed when the plants weretreated with specific ratios of ABA and malic acid. See Tables 15-18,below. Yield is presented as panicle weight, where grain yield isabout >95% of the panicle weight. The correlation between grain andpanicle weights was >0.99. Individual applications of ABA (30 ppm) andmalic acid (100 ppm) both decreased yield, while the mixture at a ratioof 1:3.3 unexpectedly increased yield by 8.2%. See Table 15, below.

TABLE 15 Panicle Yield % Change OCE/ECE Treatment and dose (g) from STCratio STC 7.73 n/a n/a ABA 30 ppm 7.29 −5.8% n/a Malic acid 100 ppm 5.40−30.1% n/a ABA + Malic acid 8.36 8.2% 1.7 (30 + 100 ppm)

The combination also unexpectedly improved rice yield at a ratio of 1:10ABA (30 ppm) to malic acid (300 ppm). See Table 16 below. The mixture ofABA and malic acid resulted in 3.9% higher grain yield compared to thesurfactant-treated control (STC).

TABLE 16 % Change OCE/ECE Treatment and dose n = 6 Panicle Yield fromSTC ratio STC 18.48 n/a n/a ABA 30 ppm 19.00 2.8% n/a Malic acid 300 ppm16.84 −8.9% n/a ABA + Malic acid (30 + 19.20 3.9% 1.1 300 ppm)

In a similar study, rice plants were subjected to water deficit stressduring early grain filling stages. The mixture of ABA (30 ppm) and malicacid (300 ppm) treated twice at around 10 and 17 days post-anthesisresulted in an unexpected increase in grain yield. See Table 17, below.The 1:10 ratio of ABA to malic acid mixture caused an unexpectedincrease in grain yield as compared to the compounds appliedindividually.

TABLE 17 Grain Yield % Change OCE/ECE Treatment and dose n = 7 (g) fromSTC ratio STC 11.56 n/a n/a ABA 30 ppm 11.88 2.7% n/a Malic acid 300 ppm10.58 −8.5% n/a ABA + Malic acid (30 + 12.10 4.6% 1.1 300 ppm)

In another study, an ABA (30 ppm) and malic acid (1000 ppm) mixture at aratio of 1:33.3 resulted in an unexpected increase in rice yield. SeeTable 18. The mixture showed a 7.8% increase in grain yield compared tothe surfactant-treated control.

TABLE 18 % Change OCE/ECE Treatment and dose n = 7 Panicle Yield fromSTC ratio STC 16.50 n/a n/a ABA 30 14.98 −9.2% n/a Malic acid 1000 17.274.7% n/a ABA + Malic acid (30 + 17.79 7.8% 1.1 1000 ppm)

Stomatal conductance is a measure of the rate of gas exchange at thesurface of a plant leaf. It is typically measured with a porometer usingunits of mmol m⁻² s⁻¹ vapor pressure. Following application of ABA torice plants, stomatal conductance of the flag leaves of the mainpanicle, the first tiller and second tiller of seven plants of the milkstage in grain development of the main panicle were measured. Weobserved a reduction in leaf stomatal conductance within one day ofapplication. See Table 19, below, demonstrating stomatal conductance(mmol m−2 s−1) of flag leaves of rice plants following foliar ABAapplication.

TABLE 19 Treatment One (1) day Two (2) days Treated Control 277.4 286.0S-ABA, 10 ppm 230.3 280.5 S-ABA 30 ppm 182.7 209.1

It is notable that the effect of ABA on stomatal conductance isshort-lived, particularly at a low rate of ABA. The addition of malicacid to ABA significantly increased the effects of ABA or malic acid onrice flag leaf transpiration 24 h post-application. Table 20 shows theaverage of three separate studies examining the effects of ABA, malicacid or the mixtures on flag leaves of plants during grain fill. Thedata were also subjected to a calculation for OCE/ECE ratio.

TABLE 20 % Change Transpiration compared OCE/ECE Treatment at 1 day toControl Expected ratio Treated Control 275.9 0.0% ABA, 10 ppm 277.6 0.6%ABA, 30 ppm 245.2 −11.1% Malic acid, 300 ppm 255.8 −7.3% ABA 10 ppm +238.6 −13.5% −6.7% 2.02 Malic acid, 300 ppm ABA 30 ppm + 228.5 −17.2%−18.4% 0.93 Malic acid, 300 ppm

The results clearly demonstrate that ABA and malic acid activity wasunexpectedly increased by co-application at a ratio of 1:30 (ABA:malicacid). This reduction in stomatal conductance increases water bankingand reduces stress caused by drought.

Example 11 Increased Yield in Wheat Plants Under Non-Stressed Conditions

A 10 feet by 50 feet plot were each treated at one week after anthesiswith either 2 grams per hectare (“g/HA”) ABA, 6 g/HA ABA, 20 g/HA, malicacid, 60 g/HA malic acid or a mixture thereof; chemical treatment withsame compounds was repeated one week after initial spray. Plants werewell-watered during entire period from planting to harvest. The plot washarvested to determine total yield except 3, 30 centimeter rows, whichwere harvested for detailed yield component analysis (above groundbiomass (“AGB”), grain yield, harvest index, above ground moisturecontent (“AG Moisture”), number of heads, number of seeds per head,1,000 seed weight and # of seeds. Results can be seen in Table 21,below.

TABLE 21 Grain AG Yield AGB Yield Harvest Moisture (g/m²) (g/m²) (g/m²)Index (%) STC 379 1159 324 0.28 18.4 ABA 2 g/HA 372 1121 347 0.31 20.2ABA 6 g/HA 383 1064 315 0.3 19.4 Malic acid 60 g/HA 369 1020 281 0.2819.1 ABA 2 g/HA + 431 1256 432 0.35 21.2 Malic acid 20 g/HA ABA 2 g/HA +415 1269 406 0.32 21.9 Malic acid 60 g/HA OCE/ECE Ratio* 1.1 1.4 1.5 1.11.1 1,000 # of # of Seed Weight # of heads/m² seeds/head (grams)seeds/m² STC 1041 14.3 22.3 14553 ABA 2 g/HA 1024 14.7 23.2 14909 ABA 6g/HA 943 14.6 22.5 13817 Malic acid 60 g/HA 897 14.4 22.2 12748 ABA 2g/HA + 953 19.23 23.9 18087 Malic acid 20 g/HA ABA 2 g/HA + 1087 15.722.5 17326 Malic acid 60 g/HA OCE/ECE Ratio* 1.4 1.1 1.0 1.4 “STC”denotes surfactant treated control *based on percent change from STC

As can be seen in Table 21, a mixture of ABA and malic acid at a 1:30ratio demonstrated an unexpected increase in yield, above groundbiomass, grain yield, harvest index, above ground moisture content,heads per meter squared, seeds per head and seeds per meter squared.

What is claimed is:
 1. A method of improving yield in a plant comprisingapplying an effective amount of (S)-abscisic acid (ABA) and malic acidto the plant, wherein the weight ratio of ABA to malic acid is fromabout 3.3:1 to about 1:33.3.
 2. The method of claim 1, wherein theweight ratio of ABA to malic acid is from about 3.3:1 to about 1:10. 3.The method of claim 1, wherein the plant is a monocotyledonous plant. 4.The method of claim 3, wherein the monocotyledonous plant is a grass. 5.The method of claim 4, wherein the grass is a grain crop.
 6. The methodof claim 5, wherein the grain crop is a cereal grain crop.
 7. The methodof claim 6, wherein the cereal grain crop is selected from the groupconsisting of corn, rice, and wheat.
 8. The method of claim 1, whereinthe plant is a dicotyledonous plant.
 9. The method of claim 8, whereinthe plant is selected from the group consisting of cucumber, lettuce,soybean, tomato and sunflower.